Structural Plasticity and Functional Interactions of the Signaling Adapter NHERF

项目摘要 许多参与细胞信号传导的蛋白质都含有蛋白质相互作用模块的串联重复序列 属于PDZ（PSD 95-Dlg 1-Zo 1）结构域家族，其识别短氨基酸基序， 通常但不排他地位于蛋白质的C末端。PDZ结构域的亲和力和特异性 对于典型的（C-末端）肽配体已经被广泛研究。虽然有证据表明， 分子PDZ-肽相互作用可以通过竞争性分子内相互作用来调节， 这种自身抑制作用的基础和激活机制还没有很好的理解。为了解决这个 我们关注Na+/H+交换调节因子1（NHERF;也称为NHERF 1或EBP 50）， 衔接蛋白参与调节重要的细胞信号事件在膜细胞骨架界面 上皮细胞NHERF包含两个PDZ结构域和C-末端ezrin结合基序（EBM），以及长 无序区域该项目的第一个目标是建立结构/动态之间的联系， NHERF的性质及其对生理相互作用伴侣的亲和力。基于荧光的结合测定， 快速混合和各种NMR技术，包括化学位移分析、顺磁弛豫研究 和H-D交换，将提供详细的洞察（i）NHERF识别肽的机制 来自生物相互作用伴侣的C-末端的配体，（ii）结构和热力学基础 调节PDZ结构域的结合亲和力和特异性，以及（iii）激活机制 由细胞骨架相互作用伴侣引发。第二个目的是阐明NHERF在调节 表皮生长因子受体（EGFR）是一种重要的细胞因子， 许多癌症的致癌驱动因素。细胞生物学研究确定了C-末端的潜在相互作用位点 EGFR的第一个PDZ结构域对应于NHERF的第一个PDZ结构域。然而，没有任何生物物理 体外研究，这些相互作用的结构细节和生物学意义很差 明白我们试图填补这些知识空白（i）通过使用NMR和荧光技术， 阐明NHERF和细胞质调节因子之间相互作用的结构和能量基础， 区域EGFR相互作用，（ii）膜环境和效应物影响的生物物理学研究 蛋白质对NHERF-EGFR相互作用的影响，以及（iii）通过使用流式细胞术和细胞成像方法， 探索与NHERF相互作用对EGFR内化、再循环和/或降解的影响， 人癌细胞系。这些细胞生物学方法，结合结构和机制， 从拟议的生物物理学研究中了解NHERF的激活及其与EGFR的相互作用， 将产生详细的新的洞察作用NHERF在调节稳定性，细胞定位和细胞 EGFR的生长刺激活性。

PROJECT SUMMARY Numerous proteins involved in cell signaling contain tandem repeats of protein-protein interaction modules belonging to the family of PDZ (PSD95-Dlg1-Zo1) domains, which recognize short amino acid motifs that are typically, but not exclusively, located at the C-terminus of a protein. The affinity and specificity of PDZ domains for canonical (C-terminal) peptide ligands has been studied extensively. While there is evidence that inter- molecular PDZ-peptide interactions can be modulated by competing intramolecular interactions, the structural basis of such autoinhibitory effects and the mechanism of activation are not well understood. To address this question, we focus on Na+/H+ exchanger regulatory factor 1 (NHERF; also known as NHERF1 or EBP50), an adapter protein involved in regulating important cell signaling events at the membrane-cytoskeleton interface of epithelial cells. NHERF contains two PDZ domains and a C-terminal ezrin-binding motif (EBM), as well as long disordered regions. The first aim of this project is to establish a linkage between the structural/dynamic properties of NHERF and its affinity for physiological interaction partners. Fluorescence-based binding assays, rapid mixing and various NMR techniques, including chemical shift analysis, paramagnetic relaxation studies and H-D exchange, will provide detailed insight into (i) the mechanism by which NHERF recognizes peptide ligands derived from the C-termini of biological interaction partners, (ii) the structural and thermodynamic basis of regulation of the binding affinity and specificity of the PDZ domains, and (iii) mechanisms of activation triggered by cytoskeletal interaction partners. The second aim is to elucidate the roles of NHERF in regulating signaling, endocytosis and degradation of the epidermal growth factor receptor (EGFR), an important oncogenic driver of many cancers. Cell-biological studies identified potential interaction sites in the C-terminal regulatory region of EGFR for the first PDZ domain of NHERF. However, there have been no biophysical studies in vitro, and the structural details and biological implications of these interactions are poorly understood. We seek to fill these gaps in knowledge (i) by using NMR and fluorescence techniques to elucidate the structural and energetic basis of interactions between NHERF and the cytoplasmic regulatory region EGFR interactions, (ii) biophysical studies of the influence of the membrane environment and effector proteins on NHERF-EGFR interactions, and (iii) by using flow cytometry and cellular imaging approaches to explore the impact of interactions with NHERF on internalization, recycling and/or degradation of EGFR in human cancer cell lines. These cell-biological approaches, combined with the structural and mechanistic insight into activation of NHERF and its interactions with EGFR gained from the proposed biophysical studies, will yield detailed new insight into the role NHERF in regulating the stability, cellular localization and cell growth-stimulating activities of EGFR.